Multiplex automated genome engineering (MAGE) uses short oligonucleotides to scarlessly modify genomes; however, insertions >10 bases are still inefficient but can be improved substantially by selection of highly modified chromosomes. Here we describe 'coselection' MAGE (CoS-MAGE) to optimize biosynthesis of aromatic amino acid derivatives by combinatorially inserting multiple T7 promoters simultaneously into 12 genomic operons. Promoter libraries can be quickly generated to study gain-of-function epistatic interactions in gene networks.
Bibliographical noteFunding Information:
We thank J. Keasling, A. Juminaga, P. Carr, S. Kosuri, J. Aach and T. Gianoulis for helpful discussions and data interpretation, H. Salis (Pennsylvania State University) for providing pJ401, and C. Voigt (Massachusetts Institute of Technology) for providing pN249. This work was funded by multiple programs from the US National Science Foundation (SynBERC, SA5283-11210), the Department of Energy (Genome to Life Center, DE-FG02-03ER6344) and the Wyss Institute for Biologically Inspired Engineering. Additional funding was from the Next-Generation BioGreen 21 Program (SSAC, PJ008109), Rural Development Administration and the Intelligent Synthetic Biology Center of Global Frontier Project funded by the Ministry of Education, Science and Technology (2011-0031956), Korea. H.H.W. is supported by the Wyss Institute Technology Development Fellowship and the US National Institutes of Health Director’s Early Independence Award (grant DP5OD009172). J.J. is supported by the National Junior research fellowship from the National Research Foundation of Korea (2012-0000391).
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology